Immunoprecipitation Protocol

Immunoprecipitation Protocol

This article describes the immunoprecipitation protocol:

Sample Preclearing

  1. Wash protein A- or G-Sepharose or protein L-Agarose with 10X volume of RIPA buffer.
  2. Vortex and centrifuge for 1 minute in a microfuge.
  3. Resuspend the pellet in the original volume that the protein A or protein G matrix was in.
  4. Add protein A- or G-Sepharose or protein L-Agarose to the sample and incubate at 4°C for 30 minutes with shaking.
  5. Centrifuge for 1 minute in a microfuge to pellet absorbed nonspecific proteins and insoluble material. Discard.

Incubation with Primary Antibody

  1. Add primary antibody to precleared sample and incubate for 1 hour at 4°C with gentle agitation.
  2. Add protein-A or -G Sepharose beads and incubate 1 hour at 4° C with gentle agitation.
  3. Centrifuge for 1 minute in a microfuge. Wash the pellet in 1 ml of RIPA buffer.
  4. Repeat twice.

Removal of Antibody-antigen Complex

  1. Resuspend immunoprecipitate in a buffered solution with either 1% SDS and 15 mM beta-mercaptoethanol or 8M urea.
  2. Heat at 90-100°C for 5-20 minutes with occasional vortexing.
  3. Centrifuge for 1-2 minutes in a microfuge.
  4. The supernatant is ready to be analyzed by Western blot.



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